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PubMed1993

Z-DNA structure of a modified DNA hexamer at 1.4-A resolution: aminohexyl-5'-d(pCpGp[br5C]pGpCpG).

Oligonucleotides with modification at the 5'-end have been used for various biochemical applications. As a first step to better assess the effects of those modifications on DNA conformation, we determined at 1.4-A resolution the left-handed Z-DNA structure of a DNA hexamer, aminohexyl-5'-d(pCpGp[br5C]pGpCpG), by X-ray diffraction analysis. This hexamer was crystallized in the monoclinic C2 (a = 51.13 A, b = 18.44 A, c = 34.67 A, and beta = 120.9 degrees) space group. Its structure has been refined by the restrained least-squares refinement to a final R factor of 0.164 using 3727 [> 2.0 sigma (F)] observed reflections. The overall conformation of the double helix resembles that of the canonical Z-DNA. The terminal 5'-phosphate groups of the dC residues adopt conformations (beta approximately 180 degrees and gamma approximately 60 degrees) similar to phosphodiester's conformation of the internal dC residues. Two types of interhelical stackings are observed, one of which may serve as a model for a single-strand nick in the backbone of DNA double helix. A barium ion is found to bridge two side-by-side Z-DNA helices by coordinating to the O6 and N7 atoms of two guanines simultaneously. This "cross-linking" ability of barium ion may be a useful property in promoting the reversible aggregation of nucleic acids.

Barium↗
PubMed2005

Protein-protein interactions and posttranslational modifications in mammalian base excision repair.

Base excision repair (BER) averts the cytotoxic and mutagenic effects of most endogenously produced DNA damage, including lesions that arise spontaneously due to the intrinsic instability of DNA or modifications that are formed from reactions with intracellular chemicals, such as reactive oxygen species and alkylating agents. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. In its most simplistic form, BER can be fully reconstituted with a minimum of four human proteins and is completed in just five sequential steps: (i) excision of an inappropriate base by a DNA glycosylase (e.g., uracil DNA glycosylase); (ii) incision of the DNA backbone immediately adjacent to the resulting abasic site by apurinic/apyrimidimic endonuclease 1; (iii) removal of the 5'-abasic terminal fragment, and (iv) repair synthesis to fill the gap by DNA polymerase beta; and (v) ligation to seal the remaining nick by DNA ligase 1 or a complex of DNA ligase 3 and X-ray repair cross-complementing 1. However, BER can involve the participation of other proteins as well, such as alternative DNA polymerases or one of several nonessential "auxiliary" factors. In addition, BER operates most efficiently when specific protein-protein coordination occurs. Furthermore, several BER protein activities have been shown to be regulated by posttranslational modification, and some of the physical protein interactions link BER to other DNA transaction pathways. In this review, we summarize the current state of the emerging complexities of mammalian BER, focusing on the growing number of reported protein-protein interactions and posttranslational modifications.

Animals↗
PubMed1981

Structural modification and protein recognition of DNA modified by N-2-fluorenylacetamide, its 7-iodo derivative, and by N-2-fluorenamine.

Several experimental approaches were used to compare the structural modifications introduced by the binding to DNA of N-hydroxy-N-2-fluorenamine and of N-acetoxy-N-2-fluorenylacetamide and its 7-iodo derivative. In the three binding reactions, the major part of the substitution occurred at C-8 of guanine. Formaldehyde unwinding, hydrolysis by endonuclease S(1), linear electric dichroism, circular dichroism, use of antibodies, unwinding of covalently closed circular DNA, and binding of tripeptide were thus successively examined. Most of the techniques showed marked differences between the different modified DNA's. However, some experiments failed to show discrimination between the induced structural alterations. These specific points are discussed. On the basis of all the available experimental evidence and after examination of the Cory-Pauling-Koltun molecular model, we proposed three models of DNA binding at the C-8 of the guanine residue with 1) the 7-iodo derivative (outside binding) 2), N-acetoxy-N-2-fluorenylacetamide (insertion-denaturation) and 3) N-hydroxy-N-2-fluorenamine (insertion without denaturation).

2-Acetylaminofluorene↗
PubMed1999

Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR).

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [32P]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNALysand the corresponding tRNALyscarrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNALeu(UUR)and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m2G content. Complementary sequencing of tRNALeu(UUR)has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased level of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.

Autoradiography↗
PubMed2002

High-throughput analysis of nucleic acid modification reactions using ion-pair reverse-phase high-performance liquid chromatography.

Ion-pair reverse-phase high-performance liquid chromatography is presented as a versatile platform for the rapid analysis of nucleic acid modification reactions in a high-throughput manner. This system allows both sensitive and nonradioactive assays to be developed for a variety of nucleic acid modification reactions. Examples presented here include assays for telomerase, uracil DNA glycosylase, polynucleotide kinase, T4 DNA ligase, C5-DNA methyltransferases, and the mismatch endonuclease CEL I. However, this approach is not confined to these reactions. Indeed the ability to perform a variety of nonradioactive assays with throughput times of 10 min per sample in conjunction with automated data analysis software represents a significant improvement in analytical and preparative nucleic acid enzymology.

Chromatography, High Pressure Liquid↗
PubMed1986

DNA modified by platinum derivatives cannot adopt the A-form.

DNA fragments from chicken erythrocytes were modified by cis-diamminedichloroplatinum(II), its trans-isomer and chlorodiethylenetriaminoplatinum(II) chloride. The conformation of the modified DNA fragments in ethanolic solutions was studied by circular dichroism spectroscopy. Non-modified DNA adopted the A-form in 81% ethanol. The modification of DNA by the three platinum compounds inhibited the B to A transition of DNA induced by high concentrations of ethanol roughly to the same extent. The results support the view that the binding of the platinum complexes to B-DNA lowers the conformational freedom of DNA so that it cannot acquire the A-conformation.

Animals↗
PubMed2006

Characterization of mimivirus NAD+-dependent DNA ligase.

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.

Acanthamoeba↗
PubMed2001

Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair.

DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1974

Mutations that alter the transmission of chloroplast genes in Chlamydomonas.

Two mutations are described that alter the pattern of inheritance of chloroplast genes in Chlamydomonas. The mutant gene mat-1 linked to the mating type allele mt(-) greatly increases the frequency of exceptional zygotes, i.e., zygotes that transmit chloroplast genes from the mt(-) (male) parent. In some crosses, 80-90% of the zygotes are biparental, transmitting chloroplast genes from both parents. The mat-2 mutation, linked to mt(+), acts to decrease the frequency of exceptional zygotes below the spontaneous level. The mutant effects are discussed in terms of a DNA modification-restriction system, postulated to regulate the transmission of chloroplast DNA in zygotes.

Cell Nucleus↗
PubMed1998

Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.

Alanine↗
PubMed2002

DNA methylation: a profile of methods and applications.

Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene silencing in disease. There is currently a wide range of methods designed to yield quantitative and qualitative information on genomic DNA methylation. The earliest approaches were concentrated on the study of overall levels of methylcytosine, but more recent efforts havefocused on the study ofthe methylation status of specific DNA sequences. Particularly, optimization of the methods based on bisulfite modification of DNA permits the analysis of limited CpGs in restriction enzyme sites (e.g., combined bisulfite restriction analyses and methylation-sensitive single nucleotide primer extension) and the overall characterization based on differential methylation states (e.g., methylation-specific PCR, MethyLight, and methylation-sensitive single-stranded conformational polymorphism) and allows very specific patterns of methylation to be revealed (bisulfite DNA sequencing). In addition, novel methods designed to search for new methylcytosine hot spots have yielded further data without requiring prior knowledge of the DNA sequence. We hope this review will be a valuable tool in selecting the best techniques to address particular questions concerning the cytosine methylation status of genomic DNA.

5-Methylcytosine↗
PubMedDate not supplied

[The effect of hydrogen ions on the B-A transition in DNA].

The effects of hydrogen ions binding to DNA on its secondary structure and B to A transition were studied by methods of X-ray diffraction and infrared spectroscopy. Helical parameters of DNA molecules with different degrees of protonation were determined. It was shown that H+-ions binding stabilize the B form of DNA in fibers in the wide range of water and inorganic salt content. Only 0.03 H+-ions bound to each nucleotide are sufficient to prevent B to A transition caused by a relative humidity decrease in DNA fibers, containing 4% of NaCl. The effective stabilization of the DNA B form by H+-ions binding is explained by modifications in DNA - solvent molecules interactions, especially in the major groove of double helices.

Animals↗
PubMed1995

Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic acid and cofactor analogues.

The DNA-binding properties of the EcoRV restriction endonuclease and modification methyltransferase with their recognition sequence (GATATC) were analyzed using the electrophoretic band-shift assay. It has previously been observed that the endonuclease does not bind specifically to GATATC sequences in the absence of the essential cofactor Mg2+. To investigate any possible roles for Mg2+ in promoting specific DNA binding, a set of hydrolysis-resistant oligonucleotide substrates were synthesized that contained either phosphate (phosphorothioate, 3'-S-phosphorothiolate), sugar (4'-thiothymidine), or base (7-deaza-2'-deoxyadenosine) modifications. However, it was found that none of these were specifically bound by the endonuclease in either the absence or the presence of Mg2+. In contrast, the methylase bound to GATATC sequences much more strongly than to nonspecific sites, and it was possible to observe the formation of enzyme--DNA complexes by gel retardation. Binding to GATATC sequences was increased by the addition of sinefungin, a nonreactive analogue of the essential cofactor S-adenosyl-L-methionine (AdoMet). Presumably this also occurs with AdoMet although methylation and turnover prevented its direct observation. In the presence of sinefungin the strongest binding was observed with hemimethylated EcoRV sequences (Kd = 11-13 nM), and unmethylated DNA was bound less well (Kd = 46 nM). Specific, albeit weaker binding was also seen with the dimethylated product (Kd = 143 nM). A difference in electrophoretic mobility was observed between enzyme-substrate and enzyme-product complexes suggestive of structural differences between them. The Kapp value found for sinefungin, with the hemimethylated EcoRV sequence, was 10.9 mM.

Adenosine↗
PubMed2006

Structure and function of protein modules in chromatin biology.

Chromatin-mediated gene transcription or silencing is a dynamic process in which binding of various proteins or protein complexes can displace nucleosomal histones from DNA to relieve repression or drive the gene into a highly repressed, silent state. Covalent modifications to DNA and histones associated with chromatin structural change play a crucial role in transcriptional regulation, with particular modifications on certain residues associated with a specific transcriptional outcome. In recent years a number of structural domains have been identified within chromatin-associated proteins, including DNA or RNA binding domains, protein-protein interaction domains and domains that recognize specific covalent modifications to histone tails. In this review we discuss the structural features of these protein modules and the functional roles they play in chromatin biology.

Acetylation↗
PubMedDate not supplied

An experimental selection system to identify bacterial cells exhibiting a new DNA host specificity.

Restriction-modification enzymes interact with DNA sequences in a highly specific manner. Mutations within the DNA binding region of the enzymes could be expected to produce enzyme variants with changed DNA sequence specificities. We developed an efficient in vivo selection system that enabled us to detect one cell coding for a restriction-modification system with a new DNA sequence specificity in a background of more than 10(6) cells with the original DNA sequence specificity.

Bacteriophage lambda↗
PubMed2002

Restriction enzyme recognition sequence search program.

A critical and difficult part of characterizing restriction enzymes and methylases is the identification of recognition sequences. To simplify this process, we have developed a plasmid transformation method along with a computer program named RM search that determines the exact recognition sequences for given restriction and modification systems.

Algorithms↗
PubMed1988

DNA adducts formed by the comutagens harman and norharman in various tissues of mice.

Covalent modifications of DNA in various tissues of mice with harman or norharman were analyzed by 32P-postlabeling assay. Administration of 0.1% harman to mice in their diet for 4 weeks resulted in DNA adducts in the liver and kidney. No specific DNA adduct was detected in other tissues, such as the glandular stomach, large intestine and brain. Similar treatment of mice with norharman resulted in DNA adducts in the kidney, glandular stomach and large intestine, but not in the liver or brain. These results suggests the in vivo genotoxicities of harman and norharman.

Alkaloids↗
PubMed1986

Inhibitors of Bacillus subtilis DNA polymerase III. Influence of modifications in the pyrimidine ring of anilino- and (benzylamino)pyrimidines.

Substituent effects governing inhibition of DNA polymerase III from Bacillus subtilis were examined in several series of N6-substituted 6-aminopyrimidines. The presence of alkyl groups as large as n-butyl in the 3-position of 6-(5-indanylamino)uracil had no effect on inhibitor-enzyme binding. Substituents in the 4-position of a series of 2-amino-6-(benzylamino)pyrimidines had complex effects: alkoxy and phenoxy derivatives were less active than the parent 4-oxo (isocytosine) compound, but alkylphenoxy and halophenoxy derivatives were more active than the 4-phenoxy compound itself, suggesting that hydrophobic binding can occur between 4-substitutents and the enzyme surface and that space between the pyrimidine ring and pol III may represent the active site of the enzyme. Replacement of 5-H by methyl and ethyl groups drastically decreased inhibitory activity of 6-(benzylamino)- and 6-p-toluidinouracils, but 5-bromo and 5-iodo analogues were equipotent with the parent compounds. These results indicate that the phenyl rings of these compounds must exist in conformations in which they are perpendicular to the pyrimidine ring plane and that charge-transfer stabilization of such "active conformations" may compensate for steric barriers from 5-halo groups in the inhibitor-enzyme complex.

Amines↗